胆汁酸G-蛋白偶联受体通过p38MAPK通路诱导-第三军医大学学报.DOCVIP

胆汁酸 G-蛋白偶联受体通过 p38 MAPK 通路诱导炎症细胞IL-1β、TNFα和IL-6的表达 张闻宇1，2，黄文栋2，娄桂予2，3 （1.河南省郑州人民医院内分泌科；2.美国City of Hope医学中心Beckman研究所；3.第三军医大学基础部生物化学与分子生物学教研室） [摘要] 目的 探讨胆汁酸G-蛋白偶联受体（TGR5）被Oleanolic acid（OA）激活后对RAW264. 7 细胞IL-1β、TNFα和IL-6表达的影响及相关机制。 方法 通过实时荧光定量（Real-time）PCR 法检测OA作用不同剂量、不同时间RAW264. 7 细胞和原代枯否细胞IL-1β、TNFα和IL-6 mRNA的表达；并进一步分析在RAW264. 7 细胞中加入三种不同信号通路的抑制剂对上述三种炎症因子表达的影响。OA作用RAW264. 7 细胞和原代枯否细胞不同时间后，Western blot 分析p38 MAPK 的磷酸化水平。 结果 OA刺激RAW264. 7细胞6h、12h、24h后，IL-1β、TNFα和IL-6mRNA表达明显升高，并呈剂量依赖关系。OA作用于分离的原代枯否细胞 3h 和 6h 后，也可观察到相同的结果。p38 MAPK 特异性抑制剂SB203580可以明显地抑制OA诱导的RAW264. 7 细胞内L-1β、TNFα和IL-6 mRNA的表达，但PKA和NF-κB的抑制剂无此作用。RAW264. 7 细胞和原代枯否细胞经OA刺激后，p38 MAPK磷酸化水平明显增强。 结论 TGR5可能通过活化 p38 MAPK 磷酸化诱导炎症细胞 IL-1β、TNFα和IL-6的表达，提示TGR5在无其他刺激因素作用下，具有诱导炎症因子表达的作用。 [关键词] 胆汁酸G-蛋白偶联受体；Oleanolic acid；RAW264. 7细胞株；枯否细胞；促炎因子[基金项目] 国家自然科学基金（ [基金项目] 国家自然科学基金 [通信作者] 娄桂予，电话：（023E-mail: louguiyu@. TGR5 induces IL-1β, TNFα and IL-6 expression by p38 MAPK pathway in macrophages Zhang Wenyu1,2, Huang Wendong2, Lou Guiyu2,3 (1.Department of Endocrinology, The People’s Hospital of Zhengzhou, Zhengzhou, 450003,China,; 2. Backman research institution, City of Hope, Duarte, U.S.A, 3. Department of Biochemistry, [Abstract] Objective To investigate the effect and mechanism of plasma membrane-bound G protein-coupled receptor for bile acids (TGR5) activation by Oleanolic acid (OA) on the expression of IL-1β, TNFα and IL-6 in macrophages. Methods Real-time PCR was used to detect the expression of IL-1β, TNFα and IL-6 in mRNA level after RAW264.7 cells and Kuffer cells were incubated with OA in different time and dose. And the expression of these inflammatory factors were further analyzed used the same method when RAW264.7 cells were stimulated by inhibitors of three different signal pathway plus OA. The phosphorylation level of p38 MAPK was measured by Western blot. Results Treatment of RAW264.7 cells with OA resulted in a robust increase in IL-1β, IL-6 and TNF-α transcripts at 6, 12 a